HUVECs (ATCC) at passage 6 were cultured in complete endothelial growth medium (VascuLife VEGF kit, Lifeline) with 10% FBS (Hyclone) concentration. Culture substrates were rinsed in Dulbecco’s phosphate buffered saline (PBS), incubated in rat tail collagen-I (50μg/mL, Corning) for at least 60min, and seeded with HUVECs at a density of 4.2×104 cells/cm2. HUVECs were cultured for 24hrs and then stimulated with TNF-α (100U/mL, R&D Systems) for another 24hrs before subsequent experiments. Culture substrates U937 cells (ATCC) were cultured in RPMI 1640 media (Gibco) supplemented with 10% FBS, 1% penicillin-streptomycin, and 2mM L-glutamine in ultra-low attachment surface flasks (Corning) up to 2×106 cells/mL. All cells were incubated at 37°C and 5% CO2.
Ultra low attachment surface flasks
Manufactured by Corning
Sourced in United States
Ultra-low attachment surface flasks are designed to promote the growth and maintenance of suspended cell cultures. The flasks feature a specialized surface treatment that minimizes cell attachment, supporting the culture of cells in suspension.
Automatically generated - may contain errors
Lab products found in correlation
B27 supplement, by Thermo Fisher Scientific (2 mentions)
L glutamine, by Thermo Fisher Scientific (2 mentions)
Dmem f12, by Thermo Fisher Scientific (2 mentions)
Cellbind surface culture flasks, by Corning (2 mentions)
Rpmi 1640 media, by Thermo Fisher Scientific (1 mentions)
Rat tail collagen 1, by Corning (1 mentions)
Tnf α, by R&D Systems (1 mentions)
Otx015, by Cayman Chemical (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Iscove modified dulbecco media (imdm), by Thermo Fisher Scientific (1 mentions)
5 protocols using ultra low attachment surface flasks
1
HUVEC Activation and Immune Cell Culture
2
Culturing Pancreatic Cancer Stem Cells
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Pancreatic tumours were obtained from PDX as described previously.3 (link) Tumours were homogenised using a GentleMACS Dissociator followed by enzymatic digestion with collagenase P for 15 min at 37°C and cultured in DMEM+10% FCS. Culture under low adhesion conditions to enrich for CSCs and human pancreatic cancer xenografts have been described previously.22 (link) To culture spheres enriched in CSCs, cells were resuspended in 1× DMEM/F-12 (Gibco) supplemented with 20 ng/mL FGF-2 (CellGS), 0.4% amphotericin B, 1% penicillin/streptomycin, 2% B27 supplement (Gibco) and 200 mM of L-glutamine (Gibco). A cell suspension of 10 000 cells/mL was then prepared and distributed into ultra-low attachment surface flasks (Corning, New York, USA) for 1 week. Prior to use, spheres were filtered (40 µM).
3
Cardiosphere-Derived Cell Isolation Protocol
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Donor hearts were obtained from organ procurement organizations under an IRB-approved protocol from Cedars-Sinai Medical Center and processed as described by RR Makkar et al.7 (link) with modifications. These samples were obtained following informed consent of the donor or next of kin. All methods were carried out in accordance with relevant guidelines and regulations. A combination of atrial and septal tissue was used to seed explant fragments without previous collagenase digestion. Explants were seeded onto CellBIND surface culture flasks (Corning) for 10–21 days before harvest of explant-derived cells (EDC) and formation of cardiopsheres in ultra-low attachment surface flasks (Corning) for 3 days. CDCs were obtained by seeding cardiospheres onto fibronectin-coated dishes and cultured until passage 5. All cultures were maintained at 5% CO2, 5% O2 at 37 °C, using IMDM (GIBCO; supplemented with 20% bovine serum (Equafetal, Atlas), 0.5 μg/mL gentamycin, and 99 μM 2-mercaptoethanol).
4
Pancreatic Tumor Isolation and Culture
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Pancreatic tumors were obtained from murine pancreatic cancer models (Ela-KRAS and KPC) or patient-derived xenografts as described previously[53 (link)]. Tumors were homogenized using a gentleMACS dissociator followed by enzymatic digestion with collagenase P for 15min at 37°C, and cultured in DMEM + 10% FCS. Outgrowing epithelial clones were then further expanded to heterogeneous cancer cell cultures. Stromal cells were mechanically removed by cell scraping and cultured separately. For some experiments, we required murine stromal cell rich primary PDAC cultures and used the stroma rich CKT111 PDAC cultures with mesenchymal features. Culture under low adhesion conditions to enrich for cancer stem cells and human pancreatic cancer xenografts have been described previously [54 (link)]. In some experiments, cells were treated with the BET inhibitor OTX015 (Cayman Chemicals; 500nM). To culture spheres enriched in cancer stem cells, cells were re-suspended in 1X DMEM/F-12 (Gibco™) supplemented with 20ng/ml FGF-2 (CellGS), 0.4% Amphotericin B, 1% Penicillin/Streptomycin, 2% B27 supplement (Gibco™) and 200mM of L-glutamine (Gibco™). A cell suspension of 10,000cells/ml was then prepared and distributed into ultra-low attachment surface flasks (Corning, NY, NY) for one week. Prior to use, spheres were filtered (40 μM for human spheres, 20 μM cell strainer for murine spheres).
5
Cardiosphere-Derived Cell Isolation Protocol
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Cardiosphere-derive cells (CDCs). Donor hearts were obtained from organ procurement organizations under an IRB-approved protocol and processed as described by RR Makkar et al. (20) with modifications. A combination of atrial and septal tissue was used to seed explant fragments without previous collagenase digestion. Explants were seeded onto CellBIND surface culture flasks (Corning) for 10-21 days before harvest of explant-derived cells (EDC) and formation of cardiopsheres in ultra-low attachment surface flasks (Corning) for 3 days. CDCs were obtained by seeding cardiospheres onto fibronectin-coated dishes and cultured until passage 5. All cultures were maintained at 5% CO2, 5% O2 at 37°C, using IMDM (GIBCO; supplemented with 20% bovine serum (Equafetal, Atlas), 0.5 μg/mL gentamycin, and 99 μM 2-mercaptoethanol).
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